CN112322518B

CN112322518B - Bacillus alpinus with potassium-dissolving effect and application thereof

Info

Publication number: CN112322518B
Application number: CN202011106345.7A
Authority: CN
Inventors: 蔡燕飞; 刘晓玉; 李福艳; 王玉琪; 任宗玲; 彭子倩
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2022-02-01
Anticipated expiration: 2040-10-15
Also published as: CN112322518A

Abstract

The invention specifically discloses a strain of Bacillus alpina (Bacillus altitudinis) YC602 and application thereof, wherein the strain preservation number is GDMCC No. 61221; the invention separates and obtains a kalium-solubilizing bacterium YC602 from potato rhizosphere soil in Huizhou city, Guangdong province, and preliminarily identifies the kalium-solubilizing bacterium YC as bacillus alpina by adopting a 16SrRNA and rpob sequence analysis method. The bacterium can dissolve insoluble potassium feldspar, improve plant seed germination and root growth, and promote plant growth. In addition, the strain has the function of producing auxin (IAA), has obvious resistance to Cu (II), Cd (II), Zn (II), Al (III) metal stress and acid stress, and has important significance in agricultural production.

Description

Bacillus alpinus with potassium-dissolving effect and application thereof

Technical Field

The invention relates to the technical field of plant growth-promoting rhizobacteria, relates to a microorganism and application thereof, and particularly relates to a Bacillus altitudinis (Bacillus altitudinis) YC602 and application thereof.

Background

The potassium element is one of three essential nutrient elements for the growth and development of crops, and the potassium deficiency can affect the growth of plants, the development of root systems, the disease and insect resistance and the like. Although the soil is rich in potassium element, 98 percent of the potassium element exists in the form of minerals such as potassium feldspar, mica and the like, and only quick-acting potassium which does not exceed 2 percent of the total potassium content of the soil is in a form which can be directly absorbed and utilized by plants. The method for dissolving the mineral potassium in the soil by using the microorganisms is an economic, effective and environment-friendly measure for solving the problem of potassium deficiency of the soil, and has very important significance for developing ecological agriculture.

The potassium-dissolving microorganism can convert the indissolvable mineral potassium in the soil into a soluble state for direct absorption and utilization by plants. The distribution range of potassium-decomposing microorganisms in soil is wide, and a plurality of researchers screen out high-efficiency potassium-decomposing microorganisms from samples such as crop rhizosphere soil, rocks and the like. 9 potassium-removing bacteria with good potassium-removing effect are separated from rhizosphere soil of pear trees, crops, vegetables and the like by Sunjingfeng and the like (2017); chenyufeng et al (2015) separated 16 high-efficiency potassium-decomposing bacteria from rhizosphere soil of banana garden in Hainan province.

Currently, most of strains with potassium-solubilizing capability are reported to be Bacillus (Bacillus sp.), wherein Bacillus subtilis, Bacillus megaterium, Bacillus cereus and the like all have the potassium-solubilizing capability. Singh et al (2010) applied bacillus mucilaginosus, azotobacter and rhizobia with potassium-solubilizing ability to corn and wheat, and the results showed that the strains could improve biomass accumulation, potassium content and chlorophyll and crude protein content in plant tissues. Anjanadevi et al (2016) apply Bacillus subtilis and Bacillus megaterium as potassium-solubilizing bacteria to konjak plants, which can greatly increase the yield of konjak tubers. These results show the potential of bacillus in potassium-solubilizing, and lay a solid foundation for exploring potential novel potassium-solubilizing microbial resources.

Bacillus alpina (Bacillus altitidiis) was first isolated in 2006 by Shivaji et al in air samples at an altitude of 20-40 km. At present, the research on the bacillus alpins at home and abroad is mostly focused on the antagonism and the phosphate-releasing function of the strain. Bacillus alpinus separated from peach orchard soil by Yinxiao (2017) has antagonistic effect on germs such as Monilinia fructicola, sweet potato Rhinocephalus, Botrytis cinerea, banana discothrix and apple canker. Marprofao et al (2019) reported that the effective phosphorus concentration in inorganic phosphorus-containing fermentation broth inoculated with Geobacillus altitudinis much higher than that of the blank control solution without inoculation. In addition, the Bacillus alpine also has the functions of producing beta-1, 3-1, 4-glucanase, alkaline protease and the like (Chengmei et al, 2014; Liwen et al, 2014).

However, no report on potassium dissolution of the bacillus alpina exists so far, so that the research on the activation capacity and the growth promotion effect of the bacillus alpina on potassium feldspar has very important significance for developing novel potassium dissolution microorganism resources.

Disclosure of Invention

The first objective of the present invention is to provide a strain of Bacillus alpina (Bacillus altitidiis) YC602 which has the functions of potassium removal, heavy metal toxicity resistance, acid resistance and IAA production and can stably colonize plant roots according to the problems in the prior art.

The second purpose of the invention is to provide a biological agent prepared by the bacillus alpina YC 602.

The third purpose of the invention is to provide the bacillus alpina YC602 and the application of the biological agent thereof.

To this end, the first object of the present invention is achieved by the following technical solutions:

a strain of Bacillus altitudinis (Bacillus altitudinis) YC602 with the preservation number of GDMCC No.61221 is preserved in Guangdong province microorganism strain preservation center at 28 th 9 th 2020, and the address is No. 59 floor 5 of Jie No. 100 of Jie of Jielizhou province, Guangzhou city, and the classification name is Bacillus altitudinis.

The YC602 strain is obtained by separating and purifying rhizosphere soil of potatoes in Huizhou city, Guangdong province, belongs to gram-positive bacteria, has spores and produces biomembranes. The colony formed after 24h of culture on the nutrient agar culture medium is irregular, light yellow, easy to pick up, non-viscous and smooth in surface. 16S rRNA and rpob gene sequences are adopted for comparison, an evolutionary system tree is constructed by Mega software, YC602 is preliminarily identified as Bacillus altitudinis, and the 16S rRNA and rpob gene sequence determination results of the strain are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

The Bacillus alpina (Bacillus altitudinis) YC602 is prepared by the following steps:

(1) separation of

Taking potato soil in Huizhou city, Guangdong province as a screening soil sample, weighing 10.0g of the soil sample, filling a 250mL triangular bottle with 90mL, putting the triangular bottle into a shaking table, oscillating for 30min at 180rpm to fully disperse the soil sample, standing for 20-30s, taking 5mL of supernatant into a test tube, and heating for 10min at 90 ℃; add 100. mu.L of the solution to a 2mL centrifuge tube containing 900. mu.L of sterile water to give 10^-2Solution according to which 10 is obtained in succession^-3、10^-4A solution; take 100. mu.L of 10^-4The diluted solution is coated on a potassium-dissolving solid culture medium, is placed in a 30 ℃ biochemical incubator for 3-5 days, and is observed whether bacterial colonies are generated;

(2) purification of

And (3) streaking and purifying YC602 growing in a potassium-dissolving solid culture medium on a nutrient agar culture medium, selecting a single colony, inoculating the single colony into an LB liquid culture medium, culturing for 12 hours, storing in 25% glycerol, and storing in an ultra-low temperature refrigerator at minus 80 ℃.

Wherein: the potassium-dissolving solid culture medium comprises the following components in percentage by weight: 5.0g of glucose, 0.5g of magnesium sulfate heptahydrate, 0.1g of calcium carbonate, 0.005g of ferric trichloride, 2.0g of tricalcium phosphate, 2.0g of potassium feldspar, 18.0g of agar, 1L of distilled water and pH of 7.0-7.5; the nutrient agar culture medium comprises the following components in percentage by weight: 3.0g of beef extract, 10.0g of peptone, 10.0g of sodium chloride, 18.0g of agar, 1L of distilled water and pH 7.0-7.5; the LB liquid culture medium comprises the following components in percentage by weight: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1L of distilled water, wherein the pH value is 7.0-7.5.

The strain has the effect of dissolving the potassium feldspar by adopting a liquid shake flask and a flame photometer for determination. Culturing in liquid potassium-decomposing culture medium containing potassium feldspar for 3 days, wherein the concentration of soluble potassium released by the strain is 12.73 mg.L^-1. The strain is determined to have auxin production by adding LB containing L-tryptophan and an OD value measuring method by a spectrophotometerCapacity, concentration 3.67 mg.L^-1. In addition, YC602 is significantly resistant to cu (ii), cd (ii), zn (ii), al (iii) metal stress and acid stress.

The second purpose of the invention is realized by the following technical scheme:

a biological agent containing the fermentation liquor of the bacillus alpina YC602, wherein the number of the bacillus alpina YC602 is 1.25 multiplied by 10⁹cfu·mL^-1。

The preparation method of the microbial inoculum comprises the following steps: the-80 ℃ glycerol freeze-dried tube YC602 strain is picked up and streaked on an LB culture medium, and is cultured in a biochemical incubator at 37 ℃ overnight. Picking a single colony to an LB liquid culture medium, and culturing for 12h in a shaking table at the temperature of 37 ℃ at 180rpm to obtain a seed solution; inoculating the obtained seed liquid into a fresh LB liquid culture medium according to the inoculation amount of 1 percent, and culturing at 180rpm and 37 ℃ for 10-14h to obtain the fermentation liquid of the strain YC 602.

The third purpose of the invention is realized by the following technical scheme:

application of Bacillus alpina (Bacillus altitidiis) YC602 as soil potassium activator.

Application of Bacillus alpina (Bacillus altitidiis) YC602 as an IAA production promoter.

Application of Bacillus alpina (Bacillus altitidiis) YC602 as a soil heavy metal restoration agent.

The application of the Bacillus alpins YC602 as a seed germination promoter adopts Huajintian No. 1 corn seeds, and comprises two treatment groups, namely a CK group (without adding bacterial liquid) and a YC602 group, wherein each group treats 50 seeds. YC602 group 5mL of bacterial suspension and potassium feldspar (10)⁶cfu·mL^-1) Adding into a filter paper sheet containing corn seeds, adding equal amount of sterile water and potassium feldspar into the CK group, and culturing in a biochemical incubator at 25 ℃ for 7 d. After 7 days, the germination rate, plant height and root length of the seeds were measured. The results show that the germination rate and the root length of the corn seeds are respectively improved by 38.9 percent and 9.9 percent compared with the control.

The application of Bacillus alpina (Bacillus altitidiis) YC602 as a corn growth promoter is characterized in that a pot experiment is provided with two treatment groups, namely a CK group and a YC602 bacterial liquid group, wherein 15 corn seedlings are treated in each pot, 3 corn seedlings are treated in each pot, and the pot number is 5. And the YC602 bacterial suspension and the potassium feldspar are added to the root of the corn when the corn is transplanted by the bacterial liquid adding group, and the same amount of sterile water and the potassium feldspar are added to the CK group to be used as a control. After 7 days, the plant height, stem thickness, root length, fresh weight and leaf number of each treated maize were determined. The results of the corn pot experiment show that the plant height, the leaf number, the stem thickness, the fresh weight, the dry weight and the root length of the corn are respectively improved by 9.7 percent, 5.7 percent, 3.0 percent, 5.1 percent, 25 percent and 1.4 percent compared with the control.

The application of Bacillus alpina (Bacillus altitidiis) YC602 as a peanut growth promoter is characterized in that a pot experiment is provided with two treatment groups, namely a CK group and a YC602 bacterial liquid group, wherein 15 peanut seedlings are treated in each pot, 3 peanut seedlings are treated in each pot, and the pot number is 5. And adding YC602 bacterial suspension and potassium feldspar to the root of the peanut during peanut transplanting by the bacterial liquid adding group, and adding equivalent sterile water and potassium feldspar for comparison by the CK group. After 7 days, the plant height, stem thickness, root length, fresh weight and leaf number of each treated peanut were determined. The peanut pot experiment result shows that compared with CK, the plant height, branch number, dry weight, fresh weight and root length of the peanut applied with YC602 are respectively increased by 16.15%, 12.80%, 15.96%, 24.71% and 24.19%.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens out a strain of Bacillus altitudinis alpine Bacillus YC 602. The strain can dissolve insoluble potassium minerals, has the capabilities of producing IAA, resisting heavy metals and resisting acid, and effectively promotes seed germination and plant growth. The invention provides a new strain resource for microbial fertilizer by exploiting the functions of the bacillus alpina in the aspects of soil nutrient activation, heavy metal restoration and IAA production for the first time. The concrete expression is as follows:

(1) the bacillus alpina YC602 provided by the invention has potassium-dissolving capacity, and the concentration of soluble potassium reaches 12.73 mg.L^-1. The strain is determined to have the ability to produce auxin by adding LB containing L-tryptophan and a spectrophotometry OD value method, and the concentration is 3.67 mg.L^-1. In addition, YC602 is significantly resistant to cu (ii), cd (ii), zn (ii), al (iii) metal stress and acid stress.

(2) The bacillus alpina YC602 provided by the invention can promote the germination of corn seeds, and the germination rate and the root length of the corn seeds are respectively improved by 38.9% and 9.9% compared with a control.

(3) The bacillus alpina YC602 provided by the invention can obviously promote the growth of corn plants, and the plant height, leaf number, stem thickness, fresh weight, dry weight and root length of corn applied with the YC602 are respectively improved by 9.7%, 5.7%, 3.0%, 5.1%, 25% and 1.4% compared with those of a control.

(4) The bacillus alpina YC602 provided by the invention can obviously promote the growth of peanut plants, and compared with CK, the plant height, branch number, dry weight, fresh weight and root length of peanuts applying YC602 are respectively improved by 16.15%, 12.80%, 15.96%, 24.71% and 24.19%.

Drawings

FIG. 1(a) shows the gram-stained YC602, with bluish-purple bacteria; (b) as a result of spore staining, the spores were green, and the cells were red.

Figure 2 is the result of YC602 biofilm formation on solid LBGM plates.

Fig. 3 is the result of YC602 biofilm formation in liquid LBGM.

FIG. 4 is a diagram of YC602 phylogenetic tree, in which a is 16SrDNA developmental tree and b is rpob developmental tree.

FIG. 5 is a comparison of water-soluble potassium concentrations in culture solutions of different strains.

Fig. 6 shows the result of erosion of potassium feldspar by YC602 under an electron microscope.

FIG. 7 shows the results of the heavy metal and strong acid stress test of YC 602.

FIG. 8 is a graph of the results of the IAA test conducted on YC 602.

FIG. 9 is a comparison of the data for YC602 and NCIB3610 motility and peristalsis.

FIG. 10 is a comparison of the peristaltic results of YC602 and NCIB 3610.

FIG. 11 is a graph showing the results of the YC602 hemolysis test.

FIG. 12 is a graph of experimental results and data statistics for YC602 for improving maize seed germination and root growth.

FIG. 13 is a photograph of a potted corn test conducted on YC602 and a data statistical chart showing the respective indices of corn after 15 days.

FIG. 14 is a photograph of a potted peanut test conducted in YC602 and a data statistical chart of various peanut indices after 30 days.

Detailed Description

The following detailed description of the invention is to be construed as exemplary only and does not limit the invention to the specific embodiments disclosed, but rather the invention is to be construed as limited in scope by any and all modifications that fall within the true spirit and scope of the appended claims.

The following examples are given by way of routine experimentation and practice in the art, unless otherwise indicated.

EXAMPLE 1 separation and purification of YC602

(1) Separation of

Preparing a potassium-dissolving solid culture medium: 5.0g of glucose, 0.5g of magnesium sulfate heptahydrate, 0.1g of calcium carbonate, 0.005g of ferric trichloride, 2.0g of tricalcium phosphate, 2.0g of potassium feldspar, 18.0g of agar and 1L of distilled water, wherein the pH value is 7.0-7.5.

Taking potato soil in Huizhou city, Guangdong province as a screening soil sample, accurately weighing 10.0g of the soil sample, putting the soil sample into a 250mL triangular flask (containing 5-7 glass beads) filled with 90mL sterile water, putting the flask into a shaking table, oscillating for 30min at 180rpm to fully disperse the soil sample, standing for 20-30s, taking 5mL supernatant into a test tube, and heating for 10min at 90 ℃. Add 100. mu.L of the solution to a 2mL centrifuge tube containing 900. mu.L of sterile water to give 10^-2Solution according to which 10 is obtained in succession^-3、10^-4And (3) solution. Take 100. mu.L of 10^-4The diluted solution is coated on a potassium-dissolving solid culture medium and is put in a biochemical incubator at 30 ℃ for 3 to 5 days. And observing whether colonies are generated.

(2) Purification of

Nutrient agar medium: 3.0g of beef extract, 10.0g of peptone, 10.0g of sodium chloride, 18.0g of agar and 1L of distilled water, wherein the pH value is 7.0-7.5.

LB liquid medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1L of distilled water, wherein the pH value is 7.0-7.5.

Example 2 YC602 characterization

(1) Morphological characteristics of colonies

The colony formed after 24 hours of culture on the nutrient agar culture medium is irregular, faint yellow, smooth in surface, non-sticky, easy to pick up and capable of generating a biological membrane.

(2) Growth characteristics

Culturing in LB liquid culture medium at 37 deg.C and 180 deg.C for 18h to obtain viable count of 3.2 × 10⁷cfu·mL^-1。

(3) Gram stain

Selecting single YC602 colony to be cultured in a liquid LB culture medium for 12h, sucking 10 mu L of sterile water on a glass slide, sucking 5 mu L of bacterial liquid into the added sterile water, uniformly mixing, and drying and fixing on flame; primarily dyeing crystal violet for 1min, washing with water, and naturally drying; mordanting iodine solution for 1min, washing with water, and air drying; decolorizing with 95% ethanol for 30s, washing with water, and air drying; counterstaining with 0.5% Sahuang counterstaining solution for 1min, washing with water, air drying, observing under 100 times of oil microscope to obtain bluish purple thallus, as shown in FIG. 1(a), indicating that YC602 is gram-positive bacteria.

(4) Spore staining

Colonies formed after culturing for 24h on nutrient agar medium are selected and inoculated in DSM sporulation liquid medium, and cultured for 12h at 37 ℃ and 180 rpm. Mixing 1 μ L of the bacterial solution and 20 μ L of sterile water on a glass slide, coating, naturally drying, fixing by flame heating, dripping 5% malachite green staining solution, heating, steaming for 3-4 times within 30s, cooling, and washing with tap water for 30 s. Adding 0.5% of a yellow-sand compound dyeing solution, washing for 3s, drying, and observing the result by using a microscope. The results are shown in FIG. 1(b), in which spores are green and thalli are red, indicating that YC602 has spore-forming ability.

(5) Capability of producing biological film

A single colony on an LB plate was picked 602, inoculated into an LB liquid medium, and cultured at 37 ℃ for 12 hours at 180 rpm. Transferring the bacterial liquid to fresh LB liquidIn the culture medium, culturing to OD₆₀₀0.4. 0.5. mu.L of the bacterial suspension was aspirated and inoculated into liquid LBGM medium and LBGM plate containing 1.5% agar, respectively, and cultured in an incubator at 30 ℃. The ability of YC602 to produce biofilm was recorded by taking pictures at 12h, 24h, 48h, 3d, respectively. Solid LBGM results see fig. 2, YC602 produced biofilms on LBGM plates with uniform wrinkles, evident in thickness over time; control NCIB3610 produced more biofilm structure on LBGM plates, with fine lines and packed into folds, which gradually thickened with culture time. Liquid LBGM results see fig. 3, YC602 produces biofilm in the liquid LBGM and wrinkles are not uniform, increasing in thickness over time; control NCIB3610 produced more biofilm structure on LBGM plates, with fine lines and packed into folds, which gradually thickened with culture time.

(6) Molecular biological Properties

YC602 total DNA was extracted using a kit method (purchased from Biotech, Ltd.). Bacterial 16SrDNA was PCR amplified using bacterial 16SrDNA universal primer forward primer F27(AGA GTT TGA TCC TGG CTC AG) and reverse primer R1492(TAC GGC TAC CTT GTT TCACTT), 20. mu.L of PCR reaction system: DNA template 1. mu.L, forward primer F27(1mM) 0.5. mu.L, reverse primer R1492(1mM) 0.5. mu.L, ddH₂O10. mu.L, 2 XTaq PCR Mix 8. mu.L. And (3) PCR reaction system: pre-denaturation at 95 ℃ for 2min, and entering thermal cycle; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 32 cycles; extension at 72 ℃ for 10 min.

The rpob sequence of the strain was PCR amplified using the forward primer rpob F1(TGCTCGCATTAGCGAAGTGT) and the reverse primer rpob R1(ACGGC CATAGTGGGAGTAGT), as shown in SEQ ID No. 2.

20 μ L of PCR reaction system: 1 μ L of DNA template, 0.5 μ L of forward primer rpob F1(1mM), 0.5 μ L of reverse primer rpob R1(1mM), ddH₂O10. mu.L, 2 XTaq PCR Mix 8. mu.L. And (3) PCR reaction system: pre-denaturation at 95 ℃ for 2min, and entering thermal cycle; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 32 cycles; extension at 72 ℃ for 10 min.

After the PCR amplification product is recovered, sequencing is completed by Guangzhou Tianyihui gene technology limited company, the obtained DNA sequence is input into a webpage of NCBI (national center for biological information) of the United states for Blast comparison, and the sequence in a database is compared and analyzed by a Blast program, so that the result shows that the 16SrDNA sequence and the rpob sequence of the strain have higher homology with the Bacillus alpinus in GenBank, and the similarity is more than 99%.

And preliminarily identifying the Bacillus alpina (Bacillus altidinis) as the Bacillus alpina YC602 by combining the plate colony characteristics, the 16SrDNA and the rpob sequence analysis result.

Example 3 effect experiment of YC602 in dissolving mineral potassium in potassium-dissolving liquid culture medium

(1) Preparing a culture medium:

potassium-dissolving liquid culture medium: 5.0g of glucose, 0.5g of magnesium sulfate heptahydrate, 0.1g of calcium carbonate, 0.005g of ferric trichloride, 2.0g of tricalcium phosphate, 2.0g of potassium feldspar and 1L of distilled water, wherein the pH value is 7.0-7.5.

② LB liquid culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride and 1L of distilled water, wherein the pH value is 7.0-7.5.

③ LB solid culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 1L of distilled water, 18.0g of agar, and pH 7.0-7.5.

(2) Preparing a solution to be detected:

YC602 thalli in glycerol is selected and streaked on an LB solid culture medium plate, and cultured for 12h at 37 ℃. The YC602 on the plate is inoculated into a test tube containing LB liquid medium, and is cultured for 12h at 37 ℃ and 180rmp by shaking. The culture broth was transferred to a fresh LB liquid medium and shaken to OD at 37 ℃ on a shaker with 180 revolutions₆₀₀0.4. The bacterial liquid is taken out and inoculated into a 250mlL triangular flask containing 50mL of liquid potassium decomposing medium, the temperature is 30 ℃, the rotation is 180 ℃, and the shaking table culture is carried out for 3 days.

(3) Determination of Water-soluble Potassium content

Centrifuging the culture solution at 8000rpm for 5min, collecting supernatant, measuring water soluble potassium content with flame photometer, and repeating the treatment 3 times while using no inoculation as control. The results of the water-soluble potassium content change test are shown in FIG. 5. The results of fig. 1 show that: YC602 increased 12.73 mg. L in the culture compared with CK^-1Water-soluble potassium.

Example 4 Electron microscopy of the erosive Effect of YC602 on Potassium feldspar minerals

(1) Preparing fermentation liquor: will OD₆₀₀And inoculating the YC602 bacterial liquid with the concentration of 0.4 into a liquid potassium-dissolving culture medium, and culturing for 7d and 14d at the temperature of 30 ℃ to obtain YC602 fermentation liquid.

(2) Pre-processing on a machine:

firstly, fixing: preparing 2.5% glutaraldehyde solution with buffer solution system of pH6.8-7.2 to fix the sample, fixing in refrigerator at 4 deg.C for more than 3 hr;

cleaning: washing the sample and the sample bottle with phosphate buffer solution for 3 times, each time for 15-30 min;

thirdly, dewatering: dehydration with an alcohol gradient, typically set as: 30%, 50%, 70%, 90%, 100%, each concentration gradient is processed for 15-20 min;

and (4) spotting: dripping the dehydrated sample on a cover glass with proper size;

fifth, sample sticking: pasting the dried samples on a sample table according to the sequence;

sixthly, spraying gold: carrying out gold spraying on the sample for about 1min by adopting an ion sputtering instrument;

and (c) machining a machine: the sample was observed on a stage of a scanning electron microscope S3499N.

(3) The experimental result is shown in fig. 6, and YC602 can not only adhere to potassium feldspar, but also erode the potassium feldspar, so that a clear dissolution trace is formed on the surface of the potassium feldspar.

Example 5 Effect experiment of YC602 on heavy Metal and acid stresses

(1) Preparing a culture medium:

0.75% LB solid medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 1L of distilled water, 7.5g of agar, and pH 7.0-7.5.

② 1.5 percent LB solid culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 1L of distilled water, 15.0g of agar and pH 7.0-7.5.

(2) The YC602 colony on the plate is inoculated into a test tube containing LB liquid medium, and is cultured overnight by shaking at 37 ℃ and 180 turns. Transfer the culture solutionTo fresh LB liquid medium, shaking to OD at 37 ℃ on a 180-revolution shaker₆₀₀0.4. Adding 100 μ L of the bacterial solution into 4ml of 0.75% LB solid culture medium at 50-60 deg.C, reversing, mixing, pouring on 1.5% LB solid culture medium plate, and blowing on a clean bench for 20 min. The sterilized filter paper sheet was placed in the center of the plate, 10. mu.L of 6M HCl, 1000mM Al (III), 200mM Cu (II), 200mM Zn (II), and 50mM Cd (II) was dropped onto the filter paper sheet, respectively, and after drying by blowing, the filter paper sheet was placed in a 37 ℃ biochemical incubator for 24 hours. The size of the transparent circle was recorded using model strain NCIB3610 as a control, and the results are shown in FIG. 7.

The test result shows that the transparent ring diameter of YC602 generated under the stress of Cu (II), Zn (II), Al (III), Cd (II) and HCl is smaller than 3610, which indicates that YC602 shows obvious resistance to Cu (II), Zn (II), Al (III), Cd (II) and HCl; the test results provide a working foundation for applying the bacteria to the aspects of acid heavy metal polluted soil remediation and the like in the future.

Example 6 results of the IAA production test of YC602

(1) Qualitative determination of auxin production

YC602 on LB plate was inoculated as a single colony to a strain containing L-tryptophan (100 mg. L.)^-1) The LB liquid medium of (1), shaking-cultured at 30 ℃ and 180rpm for 1 d. mu.L of the bacterial suspension was dropped on a white ceramic plate, and 300. mu.L of Salkowski colorimetric solution (formulation: 50mL of 35% HClO) was added thereto₄+1mL 0.5mol·L^-1FeCl₃) 300 μ L of auxin (50 mg. L) is added^-1) The colorimetric solution of (3) was used as a positive control. The white ceramic plate is observed after being placed for 30min at room temperature in a dark condition, and the condition that the color turns red indicates that the auxin can be produced. The result shows (see fig. 8) that the yellow of the fermentation liquid added with the YC602 bacteria changes into orange yellow, which indicates that the YC602 has certain IAA production capability.

(2) Drawing of auxin standard curve

Firstly, accurately weighing 10mg of auxin, firstly dissolving the auxin by using a small amount of ethanol, and then using distilled water to fix the volume to 100mL (the concentration of the auxin is 100 mu g.mL)^-1) As stock solutions, the stock solutions were used to prepare 0 (blank), 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0. mu.g/mL^-1Series ofAnd the standard solution is used as a working solution (prepared at present).

Color development liquid: 0.5 mol. L^-1FeCl₃Solution 10mL, 35% HClO₄500mL, mix and shake before using, keep in dark place, 1mL test solution with 2mL color solution.

Thirdly, taking 8 (0-7) dry and clean test tubes, sequentially adding 2mL of auxin series concentration working solution, respectively adding 4mL of developing solution, carrying out dark heat preservation in a 40 ℃ incubator for 30min to accelerate the developing reaction, and then measuring OD₅₃₀An absorbance value of 530nm is taken as a horizontal coordinate, and the auxin concentration is taken as a vertical coordinate, so that a standard curve is drawn.

(3) Determination of auxin content in fermentation liquor

Measuring OD of bacterial suspension after fermentation by spectrophotometry₆₀₀Centrifuging 10000 revolutions of the bacterial suspension for 10min, taking supernatant, adding equivalent Salkowski colorimetric solution, standing in the dark for 30min to develop color, and measuring OD₅₃₀The value is obtained. And when the calculated bacteria concentration OD600 value is 1, the content of the auxin in the fermentation liquid per unit volume is calculated. The calculated IAA concentration of YC602 is 3.67 mg.L^-1。

Example 7 YC602 having swimming and peristalsis capabilities

Single colony of YC602 from LB solid plate is picked up in liquid LB, cultured overnight at 37 deg.C and 180rpm, transferred to fresh LB liquid culture medium, and shaken to OD₆₀₀mu.L of the bacterial suspension was dropped into the center of a 0.3% LBGM plate and a 0.5% LBGM plate containing 15mL, blown on a clean bench for 10min, and cultured in an incubator at 37 ℃. The 0.3% LBGM is used for measuring the swimming capacity of the strain, and the swimming diameter of the strain is measured at 3h, 3.5h, 4h and 5 h; the peristalsis capacity was determined by 0.5% LBGM, and the diameter of peristalsis was determined at 4h, 8h and 15h and photographed. The results are shown in fig. 9 and fig. 10, with no significant difference in motility between YC602 and control 3610.

Example 8 YC602 with hemolytic safety

Single colony of YC602 from LB solid plate is picked up in liquid LB, cultured overnight at 37 deg.C and 180rpm, transferred to fresh LB liquid culture medium, and shaken to OD₆₀₀0.4, 3. mu.L of the bacterial solution was added dropwise to a blood agar plate, incubated at 37 ℃ for 24 hours, and observedThe presence or absence of hemolysis was observed. The results are shown in FIG. 11, which shows that the strain YC602 is hemolytic negative.

Example 9 YC602 Effect test on promoting corn seed Germination

(1) Preparing a bacterial suspension:

activating the strain, inoculating to LB liquid culture medium, culturing overnight, inoculating to fresh LB liquid culture medium, and shaking to OD₆₀₀＝1.0；

Centrifuging the bacterial liquid in a centrifuge at 8000 revolutions for 3 min;

thirdly, the thalli is resuspended in equivalent sterile water containing 0.25g of potassium feldspar;

fourthly, the concentration of the strain used for the corn seeds is 10⁷cfu·mL^-1The bacterial suspension of (4).

(2) Seed surface disinfection treatment: soaking in 75% ethanol for 2min, washing with sterile water for 2 times, soaking in 2% sodium hypochlorite solution for 2min, washing with sterile water for 2-3 times, and air drying on sterilized filter paper.

(3) The effect comparison test was performed using YC602 and a blank control, and 2 treatment groups were divided (the first group used 10mL of YC602 suspension, and the second group used 10mL of sterile water as a blank control, indicated by CK). Each treatment was performed in 2 replicates, each replicate containing 25 corn seeds (randomly selected seeds of uniform size after surface sterilization). Placing filter paper into a culture dish by using a sterilization forceps, adding the bacterial suspension onto a filter paper sheet, placing corn seeds on the filter paper sheet, culturing for 7 days in a biochemical incubator at 25 ℃, and watering a proper amount every day, wherein the water amount of each dish is the same. After 7 days, the germination rate and root length of the seeds were measured. The test results show (see fig. 12), that YC602 can significantly promote germination and root growth of corn seeds. Compared with the control, the germination rate and the root length of the seeds are respectively improved by 38.9 percent and 9.9 percent.

Example 10 YC602 test for promoting growth of potted corn

(1) Preparing a bacterial suspension:

Centrifuging the bacterial liquid in a centrifuge at 8000 revolutions for 5 min;

suspending the thallus in equivalent sterile water;

fourthly, the concentration of the strain used for potted corn is 10 per gram of soil⁶cfu bacterial suspension.

(2) Corn seedling culture:

washing corn seeds with water for accelerating germination, soaking and preserving water by using gauze, placing the corn seeds in a biochemical incubator at 25 ℃, and moving the corn seeds into seedling raising pots with 50 holes with 1 seed in each hole when the length of the corn seeds is about 1 cm. Watering a proper amount every day, and when 3 leaves grow on the corn seedlings, transplanting the seedlings for later use.

(3) And (3) experimental design:

the effect comparison test was performed using YC602 and a blank control, and 2 treatment groups were divided in total (the first group was added YC602 bacteria + potassium feldspar, denoted by YC602, and the second group was added with an equal amount of sterilized water + potassium feldspar, denoted by CK, as a blank control), and each treatment was repeated 5 times. Each pot is filled with 0.9kg of soil, and chemical fertilizers of urea and calcium superphosphate are applied, wherein the application rates are 50mg N and 25mg P per kg of soil respectively₂O₅。

Before transplanting seedlings, selecting corn seedlings with uniform growth vigor and size, transplanting the corn seedlings into a pot, applying bacterial suspension to the rhizosphere of corn before covering soil, watering a proper amount every day, wherein the water amount in each pot is the same, and paying attention to prevent water from flowing out of the bottom of the pot so as to avoid errors caused by loss of bacterial liquid and fertilizer. Photographs were taken every 7 days after seedling transplantation (fig. 13). The corn plants were harvested on day 15 and the plant height, stem thickness, leaf number, dry weight and fresh weight of the corn plants were measured. The results show that: compared with the control, the plant height, the leaf number, the stem thickness, the fresh weight, the dry weight and the root length of the corn are respectively improved by 9.7 percent, 5.7 percent, 3.0 percent, 5.1 percent, 25 percent and 1.4 percent. The addition of the separated bacillus alpina YC602 can obviously promote the release of soil effective potassium, improve the absorption of the soil potassium element by corn and further promote the plant growth.

Example 11 YC602 test for growth promoting Effect of potted peanuts

(1) Preparing a bacterial suspension:

Centrifuging the bacterial liquid in a centrifuge at 8000rpm for 5 min;

suspending the thallus in equivalent sterile water;

fourthly, the concentration of the bacterial strain for the peanut pot culture is 10⁶cfu · of bacterial suspension per gram of soil.

(2) Peanut seedling culture:

directly placing peanut seeds into seedling raising pots with 50 holes, wherein each hole is provided with 1 seed. Watering a proper amount every day, and reserving and transplanting the peanut seedlings when the peanut seedlings grow to 5 cm.

(3) And (3) experimental design:

the effect comparison test was performed using YC602 and a blank control, and 2 treatment groups were divided in total (the first group was added YC602 bacteria + potassium feldspar, denoted by YC602, and the second group was added with an equal amount of sterilized water + potassium feldspar, denoted by CK, as a blank control), and each treatment was repeated 5 times. Each pot is filled with 0.9kg of soil, and chemical fertilizers urea and calcium superphosphate are applied, wherein the application rates are 50mg N and 25mg P per kg of soil respectively₂O₅。

Before transplanting, peanut seedlings with uniform growth and size are selected and transplanted into a pot, bacterial suspension is applied to the peanut rhizosphere before earthing, a proper amount of water is watered every day, the water amount in each pot is the same, and the water is prevented from flowing out of the bottom of the pot so as to avoid errors caused by loss of bacterial liquid and fertilizer. Photographs were taken every 7 days after seedling transplantation (fig. 14). On day 15, samples were collected and the plant height, stem thickness, branch number, dry weight and fresh weight of the peanut plants were measured. The results show that: compared with CK, the plant height, branch number, dry weight, fresh weight and root length of the peanut with YC602 are respectively increased by 16.15%, 12.80%, 15.96%, 24.71% and 24.19%. The bacillus alpina YC602 separated by the method can dissolve potassium feldspar in soil, improve the utilization rate of soil potassium, and improve the absorption of peanuts to the soil potassium element so as to promote the growth of plants.

Sequence listing

<110> southern China university of agriculture

<120> bacillus alpina with potassium-dissolving effect and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 3

<211> 1418

<212> DNA

<213> Bacillus

<400> 3

gtcgagcgga cagaagggag cttgctcccg gatgttagcg gcggacgggt gagtaacacg 60

tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggagctaat accggatagt 120

tccttgaacc gcatggttca aggatgaaag acggtttcgg ctgtcactta cagatggacc 180

cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac 240

ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300

cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360

aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgcaagag taactgcttg 420

caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 480

acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag gcggtttctt 540

aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact gggaaacttg 600

agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg 660

aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg 720

ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780

agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc tggggagtac 840

ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900

gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaaccctag 960

agatagggct ttcccttcgg ggacagagtg acaggtggtg catggttgtc gtcagctcgt 1020

gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccagca 1080

ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140

caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac agaacaaagg 1200

gctgcgagac cgcaaggttt agccaatccc acaaatctgt tctcagttcg gatcgcagtc 1260

tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1320

atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgc aacacccgaa 1380

gtcggtgagg taacctttat ggagccagcc gccgaagc 1418

<210> 2

<211> 1141

<212> DNA

<213> Bacillus

<400> 2

ccatttcggt actagttagt atggacgaca ccgccagcgc agaagctacg cacgcataag 60

cgaagtgtta gaattaccaa atctcattga aattcaaacc tcttcttatc agtggtttct 120

tgatgagggt cttagagaga tgtttcaaga tatatcccca attgaggatt ttactggtaa 180

cctttctctt gaattcattg attacagcct aggggatcct aagtatcctg tagcagaatc 240

aaaagaacgt gatgtaactt actctgctcc actaagagta aaagttcgtt taattaacaa 300

agaaactgga gaagtaaaag accaagatgt gtttatgggt gatttcccaa tcatgacaga 360

cacaggtact tttatcatta atggtgcgga acgtgtaatc gtttctcagt tagtacgttc 420

tccaagtgta tatttcagtg gtaaagtaga caaaaacggt aaaaaaggtt ttactgcgac 480

tgtcattcca aaccgtggcg catggttaga atacgaaact gatgcgaagg atgtagtcta 540

tgtacgcatc gatcgcacac gtaagttgcc ggttacggtt cttttgcgtg ctctcggctt 600

cagctctgat caagagattc ttgacctcat tggcgagaat gaatacttac gcaacacgtt 660

ggaaaaagac aatacagaga atgccgataa agcacttctc gaaatctacg agcgtcttcg 720

tcctggagag ccgccaactg ttgaaaatgc gaaaagcttg ctagactctc gcttctttga 780

tccgaagaga tatgacttag caaatgttgg acgctacaag attaataaga agcttcatat 840

taaaaataga ctattcaatc aaaaattggc tgaaacgcta gttgatcctg aaacaggtga 900

aatcctagca gaaaaaggtc aaattttaga tagaagagtg cttgataaag ttcttccata 960

cttagaaaac ggcatcggat ttagaaagct ttatccaaac ggtggcgtag tagaggatga 1020

agtagaactt caatctatca agatttatgc accaactgat caagaaggcg agcaagtgat 1080

caacgtgatt ggtaatgcat atgtagagga agctgtgaag aacatcacgc atctgacatc 1140

a 1141

Claims

1. A strain of bacillus alpine (B), (B)Bacillus altitudinis) YC602, with the preservation number: GDMCC No: 61221.

2. a biological agent having a potassium-solubilizing function, which is prepared from the Bacillus alpinus YC602 of claim 1, wherein the number of the Bacillus alpinus YC602 is 1.25 x 10⁹ cfu.mL^-1。